Project Summary/Abstract While the molecular mechanisms for the regulation of vascular tone by nitric oxide are well appreciated, the targets of nitric oxide (NO) signaling at the proteome level are incomplete. Decline in synthesis and bioavailability of NO is central to the pathogenesis of cardiovascular diseases and in age-dependent deterioration of vascular function and cardiac muscle performance. However, the signaling pathways affected by the decline in bioavailable NO during ageing remain unclear. Furthermore, several current clinical trials aim to restore the levels of NO in humans with hopes to improve cardiovascular and skeletal muscle physiological function and prevent vascular disease and disability in aged populations. Presently, the signaling pathways reestablished by pharmacological restoration of NO remain unknown. Therefore, we propose to use chemoselective, high- resolution, mass spectrometry-based proteomic technologies to identify and quantify for the first time at the organ and cellular level the two post-translational modifications protein phosphorylation and cysteine S-nitrosation, that constitute the two principle NO signaling pathways. We will resolve changes in the canonical signaling cascade, activation of soluble guanylate cyclase, production of cGMP and Ser/Thr phosphorylation of proteins and the complimentary selective S-nitrosylation of cysteine residues as a function of gender, ageing and in the setting of NO deficiency before and after restoration of bioavailable NO. Guided by preliminary data we will also investigate a novel mechanism for the regulation of the NAD-dependent protein deacetylase sirtuin 2 by NO. This regulatory function may have important cardioprotective functions. Completion of the proposed aims will provide new mechanistic insights and a framework for system-level appreciation of NO signalling in the cardiovascular system.